Radio-adaptive response and correlation of non-homologous end joining repair gene polymorphisms [XRRC5 (3R/2R/1R/0R), XRCC6(C/G) and XRCC7 (G/T)] in human peripheral blood mononuclear cells exposed to gamma radiation.

BACKGROUND: Radio-adaptive response (RAR) is transient phenomena, where cells conditioned with a small dose (priming) of ionizing radiation shows significantly reduced DNA damage with a subsequent high challenging dose. The role of DNA double strand break repair gene polymorphism in RAR is not known. In the present study attempt was made to find out the influence of NHEJ repair gene polymorphisms [a VNTR; XRCC5 (3R/2R/1R/0R); two single nucleotide polymorphisms (SNPs); XRCC6 (C/G) and XRCC7 (G/T)] with DNA damage, repair and mRNA expression in human PBMCs in dose and adaptive response studies. Genomic DNA extracted from venous blood samples of 20 random healthy donors (16 adaptive and 4 non-adaptive) and genotyping of NHEJ repair genes was carried out using PCR amplified length polymorphism. RESULTS: The dose response study revealed significant positive correlation of genotypes at XRRC5 (3R/2R/1R/0R), XRCC6(C/G) and XRCC7 (G/T) with DNA damage. Donors having genotypes with 2R allele at XRCC5 showed significant positive correlation with mRNA expression level (0R/2R: r = 0.846, P = 0.034; 1R/2R: r = 0.698, P = 0.0001 and 2R/2R: r = 0.831, P = 0.0001) for dose response. Genotypes C/C and C/G of XRCC6 showed a significant positive correlation (P = 0.0001), whereas, genotype T/T of XRCC7 showed significant negative correlation (r = - 0.376, P = 0.041) with mRNA expression. CONCLUSION: Interestingly, adaptive donors having C/G genotype of XRCC6 showed significantly higher (P < 0.05) mRNA expression level in primed cells suggesting their role in RAR. In addition, NHEJ repair gene polymorphisms play crucial role with radio-sensitivity and RAR in human PBMCs.

Dose response and adaptive response of non-homologous end joining repair genes and proteins in resting human peripheral blood mononuclear cells exposed to γ radiation.

Ionising radiation induces single-strand breaks, double-strand breaks (DSB) and base damages in human cell. DSBs are the most deleterious and if not repaired may lead to genomic instability and cell death. DSB can be repaired through non-homologous end joining (NHEJ) pathway in resting lymphocytes. In this study, NHEJ genes and proteins were studied in irradiated human peripheral blood mononuclear cells (PBMC) at resting stage. Dose-response, time point kinetics and adaptive-response studies were conducted in irradiated PBMC at various end points such as DNA damage quantitation, transcription and protein expression profile. Venous blood samples were collected from 20 random, normal and healthy donors with written informed consent. PBMC was separated and irradiated with various doses between 0.1 and 2.0 Gy ((60)CO-γ source) for dose-response study. Repair kinetics of DNA damage and time point changes in expression of genes and proteins were studied in post-irradiated PBMC at 2.0 Gy at various time points up to 240 min. Adaptive-response study was conducted with a priming dose of 0.1 Gy followed by a challenging dose of 2.0 Gy after 4-h incubation. Our results revealed that Ku70, Ku80, XLF and Ligase IV were significantly upregulated (P < 0.05) at 4-h post-irradiation at transcript and protein level. Adaptive-response study showed significantly increased expression of the proteins involved in NHEJ, suggesting their role in adaptive response in human PBMC at G0/G1, which has important implications to human health.

Transcription profile of DNA damage response genes at G0 lymphocytes exposed to gamma radiation.

Ionizing radiation induces a plethora of DNA damages in human cells which may alter the level of mRNA expression. We have analyzed mRNA expression profile of DNA damage response genes involved in G(0)/G(1) check point pathway in whole blood to assess their radio-adaptive response, if any, to gamma radiation. Blood samples were collected from twenty-five random, normal, and healthy male donors with written informed consent and irradiated at doses between 0.1 and 2.0 Gy (0.7 Gy/min). DNA strand breaks were studied using comet assay, whereas DNA double-strand breaks were visualized using γH2AX as a biomarker. Dose response if any, at transcriptional level was studied for all these dose groups at 1 and 5-h post-irradiation. Adaptive response at transcriptional level was studied at three different priming doses (0.1, 0.3, and 0.6 Gy) separately followed by a challenging dose of 2.0 Gy after 4 h. For both the experiments, total RNA was isolated from PBMCs obtained from irradiated whole blood and reverse transcribed to cDNA. The level of mRNA expression of ATM, ATR, GADD45A, CDKN1A, P53, CDK2, MDM2, and Cyclin E was studied using real-time quantitative PCR. A significant dose-dependant increase in the percentage of DNA damage in tail was observed using comet assay. Similarly, increased number of foci was observed at γH2AX with increasing dose. At transcriptional level, a significant dose-dependent up-regulation at GADD45A, CDKN1A, and P53 genes up to 1.0 Gy was observed at 5-h post-irradiation (P ≤ 0.05). Radio-adaptive response at mRNA expression level was observed at CDK2, Cyclin E, and P53, whereas ATM, ATR, GADD45A, MDM2, ATM, and ATR have not shown any radio-adaptive changes in the expression profile. DNA damage response genes involved in G(0)/G(1) checkpoint pathway has important implications in terms of radiosensitivity in vivo and changes in the transcriptional profile might throw some new insights to understand the mechanism of adaptive response.